Bottom Line:
Loss of Gbeta13F, Ggamma1, or Galphas, but not any other G protein subunit, results in prevention of post-eclosion cell death and failure of the wing expansion.However, cell death prevention alone is not sufficient to induce the expansion defect, suggesting that the failure of epithelial-mesenchymal transition is key to the folded wing phenotypes.Our results provide a comprehensive functional analysis of the heterotrimeric G protein proteome in the late stages of Drosophila wing development.

Affiliation: Department of Biology, University of Konstanz, Konstanz, Germany.

ABSTRACTDrosophila genome encodes six alpha-subunits of heterotrimeric G proteins. The Galphas alpha-subunit is involved in the post-eclosion wing maturation, which consists of the epithelial-mesenchymal transition and cell death, accompanied by unfolding of the pupal wing into the firm adult flight organ. Here we show that another alpha-subunit Galphao can specifically antagonize the Galphas activities by competing for the Gbeta13F/Ggamma1 subunits of the heterotrimeric Gs protein complex. Loss of Gbeta13F, Ggamma1, or Galphas, but not any other G protein subunit, results in prevention of post-eclosion cell death and failure of the wing expansion. However, cell death prevention alone is not sufficient to induce the expansion defect, suggesting that the failure of epithelial-mesenchymal transition is key to the folded wing phenotypes. Overactivation of Galphas with cholera toxin mimics expression of constitutively activated Galphas and promotes wing blistering due to precocious cell death. In contrast, co-overexpression of Gbeta13F and Ggamma1 does not produce wing blistering, revealing the passive role of the Gbetagamma in the Galphas-mediated activation of apoptosis, but hinting at the possible function of Gbetagamma in the epithelial-mesenchymal transition. Our results provide a comprehensive functional analysis of the heterotrimeric G protein proteome in the late stages of Drosophila wing development.

pone-0012331-g005: Prevention of apoptosis is associated with, but is not sufficient to induce, the failure of wing expansion.A–B. Wild-type wings are fully expanded and show GFP (A) or F-actin (B) staining only on the margin and along the veins, demonstrating that the adult wings are mostly dead structures. C–G. Downregulation of the Gs pathway by overexpression of Gαo (C, D) or by expression of RNAi constructs targeting Gβ13F (E), Gγ1 (F), or Gαs (G) leads to both failure of wing expansion and prevention of apoptosis, as visualized by persistence of F-actin- (D) and GFP-positive cells (C, E–G). H–J. DAPI nuclear staining. Overexpression of Gαo in aged wings leads to the DAPI staining pattern (J) characteristic of the young (ca. 1h-old, H) wild-type wings; aged wild-type wing only shows DAPI staining along the veins (I). K. Expression of the apoptosis inhibitor p35 prevents cell death throughout the wing as seen by persistence of GFP-positive cells, but does not cause the failure of wing expansion. All wings presented here are from MS1096-Gal4; UAS-GFP flies which are ≥1 day-old (except for the wing of panel (H)).

Mentions:
Clonal elimination of Gαs results in failure of the cell death in the wing [22]. Indeed, while aged flies retained live GFP- and rhodamine phalloidin-stained cells only along the veins and wing margin (Fig. 5A, B), we found that the MS1096-Gal4-driven expression of Gαo or RNAi constructs targeting Gβ13F, Gγ1, or Gαs all similarly resulted in maintenance of live cells within the wing blade of well-aged flies (Fig. 5C–G). To better resolve the remaining live cells, we performed the nuclear staining with DAPI [22], [24]. Young (ca. 1h-old) wild-type wings contain many DAPI-positive living cells (Fig. 5H), but aged wild-type wings showed DAPI staining only alone the veins (Fig. 5I). In contrast, wings of the Gαo-overexpressing flies up to six days old were still filled with DAPI-positive living cells (Fig. 5J). These data clearly show that the wing expansion failure is associated with the failure of cell death. However, is prevention of the cell death sufficient to cause the folded wing phenotype? To investigate this possibility, we expressed the baculovirus apoptosis inhibitor p35 in the entire wing under the MS1096-Gal4 control. While apoptosis was efficiently prevented, wing expansion was normal in these wings (Fig. 5K). This data agrees with the similar observations obtained when p35 was expressed using other Gal4 drivers [21], [22]. Cumulatively, our data suggest that apoptosis, being an important process during post-eclosion wing maturation, is unlikely to be the sole driving force behind wing expansion. Wing expansion seems more dependent on the epithelial-mesenchymal transition [21], [24], or perhaps requires both processes to act in concert. Elimination of the components of the heterotrimeric Gs proteins apparently leads to both the failure of epithelial-mesenchymal transition and apoptosis, leading cumulatively to the wing expansion defect.

pone-0012331-g005: Prevention of apoptosis is associated with, but is not sufficient to induce, the failure of wing expansion.A–B. Wild-type wings are fully expanded and show GFP (A) or F-actin (B) staining only on the margin and along the veins, demonstrating that the adult wings are mostly dead structures. C–G. Downregulation of the Gs pathway by overexpression of Gαo (C, D) or by expression of RNAi constructs targeting Gβ13F (E), Gγ1 (F), or Gαs (G) leads to both failure of wing expansion and prevention of apoptosis, as visualized by persistence of F-actin- (D) and GFP-positive cells (C, E–G). H–J. DAPI nuclear staining. Overexpression of Gαo in aged wings leads to the DAPI staining pattern (J) characteristic of the young (ca. 1h-old, H) wild-type wings; aged wild-type wing only shows DAPI staining along the veins (I). K. Expression of the apoptosis inhibitor p35 prevents cell death throughout the wing as seen by persistence of GFP-positive cells, but does not cause the failure of wing expansion. All wings presented here are from MS1096-Gal4; UAS-GFP flies which are ≥1 day-old (except for the wing of panel (H)).

Mentions:
Clonal elimination of Gαs results in failure of the cell death in the wing [22]. Indeed, while aged flies retained live GFP- and rhodamine phalloidin-stained cells only along the veins and wing margin (Fig. 5A, B), we found that the MS1096-Gal4-driven expression of Gαo or RNAi constructs targeting Gβ13F, Gγ1, or Gαs all similarly resulted in maintenance of live cells within the wing blade of well-aged flies (Fig. 5C–G). To better resolve the remaining live cells, we performed the nuclear staining with DAPI [22], [24]. Young (ca. 1h-old) wild-type wings contain many DAPI-positive living cells (Fig. 5H), but aged wild-type wings showed DAPI staining only alone the veins (Fig. 5I). In contrast, wings of the Gαo-overexpressing flies up to six days old were still filled with DAPI-positive living cells (Fig. 5J). These data clearly show that the wing expansion failure is associated with the failure of cell death. However, is prevention of the cell death sufficient to cause the folded wing phenotype? To investigate this possibility, we expressed the baculovirus apoptosis inhibitor p35 in the entire wing under the MS1096-Gal4 control. While apoptosis was efficiently prevented, wing expansion was normal in these wings (Fig. 5K). This data agrees with the similar observations obtained when p35 was expressed using other Gal4 drivers [21], [22]. Cumulatively, our data suggest that apoptosis, being an important process during post-eclosion wing maturation, is unlikely to be the sole driving force behind wing expansion. Wing expansion seems more dependent on the epithelial-mesenchymal transition [21], [24], or perhaps requires both processes to act in concert. Elimination of the components of the heterotrimeric Gs proteins apparently leads to both the failure of epithelial-mesenchymal transition and apoptosis, leading cumulatively to the wing expansion defect.

Bottom Line:
Loss of Gbeta13F, Ggamma1, or Galphas, but not any other G protein subunit, results in prevention of post-eclosion cell death and failure of the wing expansion.However, cell death prevention alone is not sufficient to induce the expansion defect, suggesting that the failure of epithelial-mesenchymal transition is key to the folded wing phenotypes.Our results provide a comprehensive functional analysis of the heterotrimeric G protein proteome in the late stages of Drosophila wing development.

Affiliation:
Department of Biology, University of Konstanz, Konstanz, Germany.

ABSTRACTDrosophila genome encodes six alpha-subunits of heterotrimeric G proteins. The Galphas alpha-subunit is involved in the post-eclosion wing maturation, which consists of the epithelial-mesenchymal transition and cell death, accompanied by unfolding of the pupal wing into the firm adult flight organ. Here we show that another alpha-subunit Galphao can specifically antagonize the Galphas activities by competing for the Gbeta13F/Ggamma1 subunits of the heterotrimeric Gs protein complex. Loss of Gbeta13F, Ggamma1, or Galphas, but not any other G protein subunit, results in prevention of post-eclosion cell death and failure of the wing expansion. However, cell death prevention alone is not sufficient to induce the expansion defect, suggesting that the failure of epithelial-mesenchymal transition is key to the folded wing phenotypes. Overactivation of Galphas with cholera toxin mimics expression of constitutively activated Galphas and promotes wing blistering due to precocious cell death. In contrast, co-overexpression of Gbeta13F and Ggamma1 does not produce wing blistering, revealing the passive role of the Gbetagamma in the Galphas-mediated activation of apoptosis, but hinting at the possible function of Gbetagamma in the epithelial-mesenchymal transition. Our results provide a comprehensive functional analysis of the heterotrimeric G protein proteome in the late stages of Drosophila wing development.